Volume 58, Issue 3, Pages 406-417 (May 2015) Drosophila Dicer-2 Cleavage Is Mediated by Helicase- and dsRNA Termini-Dependent States that Are Modulated by Loquacious-PD  Niladri K. Sinha, Kyle D. Trettin, P. Joseph Aruscavage, Brenda L. Bass  Molecular Cell  Volume 58, Issue 3, Pages 406-417 (May 2015) DOI: 10.1016/j.molcel.2015.03.012 Copyright © 2015 Elsevier Inc. Terms and Conditions

Molecular Cell 2015 58, 406-417DOI: (10.1016/j.molcel.2015.03.012) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure 1 Domain Organization and Purification of dmDcr-2 (A) Colored rectangles depict conserved domains. Information from Expresso structure-based alignments (Armougom et al., 2006), NCBI conserved domains (Marchler-Bauer et al., 2011), and crystallographic analyses (Kowalinski et al., 2011; Tian et al., 2014) was used in defining domain boundaries. Information is limited, so the ruler/connecting helix C-terminal boundary is arbitrary. (B) Cartoon of Dicer domains in apo state (colored as in A) based on negative stain (Lau et al., 2012) and cryo-EM (Taylor et al., 2013) studies. (C) Amino acids within motif I (shaded) of Hel1, and the tandem RNase III domains (a and b), are shown with dmDcr-2 mutations (blue) used in this study. dmDcr-2RIII had a mutation in each of the RNase III domains, while dmDcr-2RIII-K34A had all three mutations shown. Studies of related helicases show that this mutation precludes ATP binding and hydrolysis (Linder and Jankowsky, 2011). (D) Coomassie-stained SDS-PAGE of 10 μg of dmDcr-2 (WT), dmDcr-2RIII (RIII), or dmDcr-2RIII-K34A (RIII-K34A) after purification to homogeneity. M, marker proteins in kilodaltons (kDa). Molecular Cell 2015 58, 406-417DOI: (10.1016/j.molcel.2015.03.012) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure 2 Binding Affinity of dmDcr-2RIII for 106 BLT and 3′ovr dsRNA (A) Increasing dmDcr-2RIII was added to 20 pM dsRNA, labeled at the 5′ end of the sense strand (top in cartoons below lanes) with 32P, and incubated 30 min at 4°C, without (top panel) or with (middle panel) 5 mM ATP. Bottom panel assays were with 5 mM ATP and identical conditions, but dsRNA was internally labeled (sense strand, 32P), and each strand had a 2′,3′-cyclic phosphate (open triangle). PhosphorImages show free dsRNA (fastest mobility) and complexes separated by 4% PAGE (n = 3). (B) Radioactivity in gels as in (A) was quantified to generate binding isotherms. Radioactivity for dsRNAtotal and dsRNAfree was quantified to determine fraction bound. Fraction bound = 1 – (dsRNAfree/dsRNAtotal). All dsRNA of slower mobility than dsRNAfree was considered bound. Data points were fit using the Hill formalism, where fraction bound = 1/(1 + (Kdn/[P]n)). Error bars, SD (n = 3). Molecular Cell 2015 58, 406-417DOI: (10.1016/j.molcel.2015.03.012) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure 3 dsRNA Termini Dictate Reactivity of dmDcr-2WT in an ATP-Dependent Manner (A) Single-turnover cleavage assays of 106 BLT and 3′ovr dsRNA (1 nM) with dmDcr-2WT (30 nM) at 25°C in cleavage assay buffer. Sense strand was 32P-internally labeled. Products were separated by 17% denaturing PAGE, and a representative PhosphorImage is shown. ∗, major siRNA product; ns, nonspecific band; Left, marker nucleotide lengths (n ≥ 3). (B) Quantification of single-turnover assays as in (A), in the absence of ATP. Data points are mean ± SD (n = 3). All dsRNA not corresponding to dsRNAuncleaved was considered cleaved. Percent 106 dsRNA cleaved versus time was fit to the pseudo-first-order equation: y = yo + A × (1-e−kt); where A = amplitude of rate curve, yo = baseline (∼0), k = pseudo-first-order rate constant = kobs; t = time (Welker et al., 2011). (C) Same as for (A) except with 5 mM ATP (n ≥ 3). (D) Same as for (B) except with 5 mM ATP (n = 3). (E) Multiple-turnover cleavage assays were with 8 nM dmDcr-2WT and 100 nM 32P-internally labeled 106 BLT or 3′ovr dsRNA with 5 mM ATP for times indicated. Products were separated by 12% denaturing PAGE, and a representative PhosphorImage is shown. ∗, major siRNA product; ns, nonspecific band (n ≥ 3). Consistent with processivity, multiple siRNAs are produced per molecule of BLT dsRNA cleaved; at present it is unclear why the reaction plateaus at 40 min, although slow product release, or product inhibition, is possible. (F) Quantification of multiple-turnover assays with 5 mM ATP as shown in (E) with methods as in (B). Data points are mean ± SD (n = 3). (G) dmDcr-2WT (200 nM) was incubated with 106 BLT or 3′ovr dsRNA (600 nM) with 100 μM ATP in cleavage assay buffer at 25°C and ATP hydrolysis monitored by thin-layer chromatography (TLC). PhosphorImage shows a representative TLC plate (n ≥ 3). Positions of origin, ATP, and ADP are indicated. (H) Quantification of ATP hydrolysis assays as in (G). Data points are mean ± SD (n = 3). Data were fit to the pseudo-first-order equation y = yo + A × (1-e−kt); where y = product formed (ADP in μM); A = amplitude of the rate curve, yo = baseline (∼0), k = pseudo-first-order rate constant = kobs; t = time. See also Figures S1–S3. Molecular Cell 2015 58, 406-417DOI: (10.1016/j.molcel.2015.03.012) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure 4 dsRNA Termini Trigger Alternate States of dmDcr-2 in an ATP- and Helicase-Dependent Manner (A) A total of 20 μg of dmDcr-2RIII (2 μM) was incubated with or without 10 μM 106 BLT or 3′ovr dsRNA, with or without 5 mM ATP in cleavage assay buffer (20 min; 25°C), before treating with trypsin (10 nM) for indicated times. At each time point, 4 μg of dmDcr-2 from the reaction mix was quenched with an equal volume of 2× SDS-PAGE loading buffer. Products were resolved by SDS-PAGE and visualized with Coomassie brilliant blue. ∗, protease-resistant fragment of ∼116 kDa observed with BLT dsRNA and ATP and analyzed by mass spectrometry and Edman sequencing; ←, protease-resistant fragment observed with 3′ovr dsRNA and ATP; M, markers, kDa noted on left. Lanes 1–14 and lanes 15–27 (see bottom of Figure 4C) were on separate gels run concurrently for same time (n ≥ 3). (B) Same as for (A) except with dmDcr-2RIII-K34A (2 μM) (n = 3). (C) Same as for (A) except with 5 mM ATP-γS (n ≥ 3). See also Figures S3 and S4. Molecular Cell 2015 58, 406-417DOI: (10.1016/j.molcel.2015.03.012) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure 5 Characterization of Cleavage Events that Require ATP Hydrolysis (A) Single-turnover cleavage assays were with dmDcr-2WT (30 nM) and 106 BLT (lanes 1–6) or 3′ovr dsRNA (lanes 7–12) (1 nM) for 90 min at 25°C in cleavage assay buffer without (−) or with (+) 5 mM ATP, 10 mM glucose (Glu), and 1 unit hexokinase (HK). Sense strands were 5′ 32P-labeled (red dot) and blocked with 2′,3′-cyclic phosphate (open triangles). Hexokinase (1 unit) and glucose (10 mM; 0.2 micromoles) were incubated in reaction containing 5 mM ATP (20 min; 25°C) to deplete ATP before addition of dmDcr-2. Products were resolved on a 17% polyacrylamide denaturing gel; a representative PhosphorImage is shown (n = 2). ∗, 22 nt major siRNA product; ∗∗, 8 nt product. Marker nt lengths (left) with 10 nt positions based on 106 sense strand mapping (Welker et al., 2011); AH, alkaline hydrolysis. (B) Same as for (A) except with 5 mM ATP analogs with or without hexokinase (HK) and glucose (Glu) to remove contaminating ATP (n = 2). Molecular Cell 2015 58, 406-417DOI: (10.1016/j.molcel.2015.03.012) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure 6 Loqs-PD Modulates Termini-Dependent Cleavage by dmDcr-2 (A) Single-turnover cleavage assays were with dmDcr-2WT (30 nM) and 32P-internally labeled 106 BLT or 3′ovr dsRNA (1 nM), without or with Loqs-PD (30 nM) and ±5 mM ATP, in cleavage assay buffer (25°C). Equimolar amounts of dmDcr-2WT and Loqs-PD were pre-incubated (5 min; ice) prior to adding dsRNA. Products were resolved on a 12% polyacrylamide denaturing gel and a representative PhosphorImage is shown. ∗, siRNA product; left, marked nt lengths (n = 3). (B) Quantification of multiple-turnover cleavage assays with 8 nM dmDcr-2WT, 100 nM 32P-internally labeled 106 BLT or 3′ovr dsRNA, 8 nM Loqs-PD as indicated, and 5 mM ATP. Data points are mean ±SD (n = 2). (C) Quantification of ATP hydrolysis assays with 200 nM dmDcr-2WT, 200 nM Loqs-PD and 600 nM 106 BLT or 3′ovr dsRNA with 100 μM ATP in cleavage assay buffer at 25°C. ATP hydrolysis was monitored by TLC; data points, mean ± SD (n = 3). (D) dmDcr-2RIII (2 μM, 20 μg) and Loqs-PD (2 μM, 4 μg) were incubated with or without 10 μM 106 BLT or 3′ovr dsRNA, ±5 mM ATP in cleavage assay buffer (20 min; 25°C), before treating with trypsin (10 nM) for indicated times. Proteins were preincubated (5 min; ice) prior to adding dsRNA. At indicated times, 4 μg of dmDcr-2 and 0.8 μg of Loqs-PD were quenched with an equal volume of 2× SDS-PAGE loading buffer. Products were resolved by SDS-PAGE and visualized with Coomassie brilliant blue. ∗, protease resistant fragment of ∼116 kDa observed with BLT and 3′ovr dsRNA with ATP and Loqs-PD. M, markers in kDa, indicated on left. Two separate gels were run concurrently for the same time (n = 3). (E) Same as for (A) except with 32P-internally-labeled dsRNA with 2′,3′-cyclic phosphates (2′,3′ > p) on each strand (n = 3). (F) Single-turnover cleavage reactions were incubated at 25°C in cleavage assay buffer for times indicated, and contained dmDcr-2WT (30 nM), 32P-internally labeled rncs-1 (1 nM), 5 mM ATP, and Loqs-PD when indicated (30 nM). Products were resolved on a 12% polyacrylamide denaturing gel; a representative PhosphorImage is shown. ∗, siRNA product; Marked nt lengths, left (n = 4). (G) Same as for (F) except with esi-2; (n = 3). See also Figures S5 and S6. Molecular Cell 2015 58, 406-417DOI: (10.1016/j.molcel.2015.03.012) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure 7 Model for Differential Substrate Recognition and Processing by dmDcr-2 dmDcr-2 (colored as in Figure 1) is shown interacting with BLT (left) and 3′ovr (right) dsRNA. Addition of ATP induces a conformational change that is favored to different degrees, as indicated by direction of red arrows. Loqs-PD (dsRBMs and isoform specific amino acids, orange spheres, and tail, respectively) interacts with the helicase domain to stabilize the closed conformation. ATP hydrolysis is shown associated with the closed conformation. Additional details in text. Molecular Cell 2015 58, 406-417DOI: (10.1016/j.molcel.2015.03.012) Copyright © 2015 Elsevier Inc. Terms and Conditions